Bacillus thuringiensis (hereinafter, sometimes abbreviated as Bt) forms endospores like other Bacillus bacteria. The spores germinate and grow into vegetative cells in the presence of suitable nutritional components. The vegetative cells repeat cell division successively and sooner or later turn into sporangia that form endospores and crystal protein in the cells due to exhaustion of nutritional components, environmental changes, and so forth. Further, the cells are destructed to release the endospores and crystal protein.
Insects eat the spores and crystal protein Bt produces. When they reach the mesenteron in the digestive tract, the protein is dissolved under strongly alkaline conditions of the digestive juice to produce a protoxin, which then is converted by a proteolytic enzyme into an active ingredient (toxin). The active ingredient binds to a receptor in an epithelial cell of the mesenteron to injure cells in the vicinity of it. In the injured part, the digestive juice and the body fluid mix with each other to change the osmotic pressure and pH in the body. As a result, the food digesting function is disturbed, paralysis of mouth-part is caused, and the feeding action is retarded in the insect. Furthermore, the spores germinate under nutritional conditions and they invade into the hemocele of the insect as the vegetative cells propagate, thus causing blood poisoning.
Although insects may have different sensitivity depending on the species of insect, usually the feeding action ceases after several hours and the insect dies after 2 or 3 days after eating Bt. It is attributable to this phenomenon that less damage by insects' eating is observed even when some insects remain alive after Bt is used. Many synthetic insecticides act on the nerve system of insect so that vigorous convulsion or knockdown effect, paralysis or the like phenomenon is observed. However, the mechanism of the action of Bt is quite different as described above and the effect is gradually exhibited even though living insects exist after the treatment. Bt and protein having a pesticidal activity (crystalline toxic protein) produced by Bt are very useful as an environmentally safe microbial pesticides (Bt agents), in particular as insecticides for Lepidoptera insects and are practically used worldwide.
Bt is gram-positive rod cells and produces crystal protein in the spore formation stage at a late stage of logarithmic phase. The crystal protein is not converted into a protein having a pesticidal activity to cause gut paralysis and systemic paralysis before it is orally taken into the digestive tract by an insect to be subjected to alkali decomposition and enzymatic decomposition in the digestive juice. However, it does not exhibit toxicity to mammals.
The crystal proteins Bt produces are formed in the sporangium along with the spores and released to the outside of the cell together with the spores after passing the phase of the sporangium (Nature, 172, 1004, 1953). These generally constitute complex crystals such as diamond-shaped, bipyramidal, rhomboidal and so forth and are insoluble in water. They are produced one per spore at the time of spore formation and released together with spores into medium as the bacterial cell is destructed. Usually they are of a steric rhombic or orthorhombic structure and have a size on the order of 2.0 μ in the long side and 0.6 μ in the short side. Subspecies include also amorphous ones and their size varies widely. On their surface, regular stripe structures can be seen. Isolation from the medium and purification of crystal protein can be performed by use of a bilayer fractionation method, a density gradient centrifugation method or the like.
The crystal proteins are soluble in an NaOH solution having a pH 12 or more. According to the analyses by SDS-PAGE (polyacrylamide gel electrophoresis), there are observed three proteins of about 130 to 135 kDa, about 65 kDa and about 80 kDa in a bacterial strain belonging to Bacillus thuringiensis. They are generically called Cry 1 protein, Cry 2 protein, and Cry 5 protein. Furthermore, they can be separated into a plurality of proteins that have almost approximate molecular weights but partially differ from each other by a fractionation operation such as high performance liquid chromatography. That is, in the case of Cry-1 protein, it is classified into proteins Cry-1Aa, Cry1Ab and so forth.
Bt was isolated from larva of Mediterranean flour moth (Ephestia kuehniella Zeller [Pyralidae]) by Berliner, a German researcher in 1911. Since the larva of the insect ate the flour from Thuringia, the insect was named Thuringiensis. Earlier than this, Dr. Ishiwatari isolated the same bacterial species as a pathogenic bacterium to silkworm in 1901. Thus, it is understood that Bt has widely occurred in the natural world since old. For example, it is present in grain warehouses and millhouses where grain pests inhabit. Also, it is detected in wagons and cabins and so forth for transporting grains. Thus it is known that it migrates everywhere in the world. Also in Japan, its distribution in every district has been examined and many Bacillus thuringiensis strains have been isolated from the dust in the houses of silkworm farmers, the surface of plants and so forth.
The bacteria that belong to the genus Bacillus amount to 70 or more species. Those strains frequently observed worldwide include 22 strains. They are distinguished basically by the ability of spore formation and shape of spore, production of gas, production of acetylmethylcarbinol (AMC), reduction of nitrates, and assimilability of some sugars in accordance with the techniques of Thiery and Franchon. Bacillus thuringiensis (B. thuringiensis) is finally distinguished from its allied species by the presence or absence of a crystal having a pesticidal activity (“Manual of techniques in insect pathology,” L. Lacey ed., Academic Press, California, 55-77 (1997)).
The characteristics used for distinguishing Bacillus thuringiensis from other bacterial species and other species belonging to the genus Bacillus include gram-positive rod, catalase (+), spore formation (+), ovary spore, 0.9 μ or more in width of vegetative cell, production of acetylmethylcarbinol (+), facultative anaerobicity, assimilation of D-mannitol (−), and existence of crystal protein (+).
For the identification of subspecies of Bt, flagellum antigen (H-antigen) according to the serological technique by De Barjac and Bonefoi using an antibody in a rabbit serum to the flagellum of a bacterium has been employed for a long time as long as 40 years (Entomophaga 7, 5-31, 1962). This is a technique that has been widely utilized for the phylogenetic systematics of Bacillus thuringiensis. 
The pesticidal activity of the bacterial strains varies depending on subspecies and is of an extremely high specificity. For example, there have been known kurustaki, aizawai and so forth as subspecies that exhibit an activity to Lepidoptera insects and tenebrionis, japonensis and so forth as subspecies that exhibit an activity to Coleoptera insects.
However, in actuality, bacterial strains belonging to the same subspecies may differ in the spectrum of pesticidal activity depending on the strain. In the case of Bt strains that have an activity to a part of Lepidoptera insect pests, the pests have acquired a resistance thereto. In addition, few reports have been made on strains exhibiting effective activity to Coleoptera insects.
Thus, a novel Bt agent that is effective to Lepidoptera insect pests having acquired a resistance to the Bt agent is demanded. Furthermore, there is a keen demand for a Bt agent having an activity to Coleoptera insects. Among these, novel Bt agents having a pesticidal activity to larvae of Coleoptera insects, in particular larvae of scarabs, thus far reported include only Bacillus thuringiensis Serovar, japonensis strain buibui) strain (Japanese Patent Application Laid-open Nos. Hei 6-65292 and Hei 7-179) and Bacillus thuringiensis var. japonensis N141 (Japanese Patent Application Laid-open No. Hei 8-228783).